Received Date: October 19, 2015; Accepted Date: February 05, 2016; Published Date: February 08, 2016.
Citation: Zeinab, Zayed (2016) Karyotype Analysis and Protein Profile for Three Trifolium Species. J Cytol Histol 7: 401. doi:10.4172/2157-7099.1000401
Copyright: © 2016 Zeinab, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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The aim of the present investigation was cytological and protein comparison among three species within the genus Trifolium. The results revealed, Idiogram of the haploid complements of T. alexandrinum, T. refeigratum and T. repens. Besides T. refeigratum and T. repens have 16 pairs of chromosomes with a pair of satellites located at the end of their short arm in chromosome 16. T. alexandrinum has 8 pairs of chromosomes whose karyotyping formula is 2 nsm (+), 10 nsm (-) + 4 nm. The T. alexandrinum was clustering alone as well as having polymorphic bands that were different from T. refeigratum and T. repens.
Clover and cytological; T. alexandrinum; T. refeigratum; T. repens
Trifolium is one of the most important genera of the Leguminosae family in Egypt and most countries of the world [1-3]. Cytological characters, including chromosome number and karyotype analysis have been considered important tool for taxonomic and evolutionary relationships . The number, size and shape of chromosomes were used to characterize the karyotype and define taxonomic differences.
Zarco  used the intra-chromosomal and inter-chromosomal asymmetry indices (A1 and A2, respectively) to define differences among cultivars while mentioned that the cultivar with high A1 and A2 values are considered more advanced than others . In general, high A1 and A2 values are scored in cultivars with higher degrees of variation in chromosome length . These variations might be due to chromosome deletions or due to different levels of condensation and differential contraction of chromosomes as suggested by El-Nahas . Soliman et al.  in Egypt identified the karyotype formula for the Egyptian clover (Trifolium alexandrinum L.). They also, determined the somatic chromosome counts for two cultivars as 2n = 16. Karyotype analysis showed differences in chromosome morphology. They studied chromosomes nsm (+) that were observed in cv Helaly. Furthermore, they stated that the karyotype formula for Helaly multiple cut as 2 nsm (+) + 2 nsm (−) + 12 nm but it was 6 nsm (−) + 10 nm for Fahl single cut cultivar.
In addition, Chen and Pryce  compared karyotypes of 15 species of Trifolium belonging to the section Amoria, on the basis of chromosome size, centromere position, number of satellite chromosomes, and size of satellites. They found that some species having similar or indistinguishable karyotypes, while others differed from one to another by one or more cytological characters. Beside they pointed out the similarity of karyotypes of T. nigrescens, T. occidentale, T. petrisavii, and T. repens L. Sudipta et al.  found amphidiploid (allotetraploid) Trifolium repens L., diploid-like meiotic behaviour of chromosomes, with no multivalent formation, and a normal karyotype with a single pair of chromosome having a secondary constriction. They explained that these characteristics might be occurred due to by favorable genetic and cytological stability in nature, and high pollen fertility.
George et al.  found the dendrogram, resulted from the hierarchical cluster analysis of SDS-PAGE profiles of seed proteins conform, with some restrictions, to the present splitting of the genus Trifolium into the sections but not into the subsections and series. In addition, the importance of electrophoretic evidence in plant systematics has been discussed in details by many workers [11-24].
Therefore, the aim of this study was to investigate karyotype and protein profile of three species of Trifolium.
Cytology and karyotyping: Viable seeds of the three species of T. alexandrinum, T. refeigratum and T. repens were kindly obtained from the Forage Crops Research Department and Egyptian Museum as part of the Egyptian flora in 2014/2015.
Seeds were germinated and actively growing root tips were pretreated for 2-4 h in 0.002 M 8- hydroxyquinoline, fixed in 3:1 (absolute ethanol : acetic acid), hydrolyzed for 5 min in 1 N HCl at 60°C and stained in aceto-orcein according to Lacour and Chattopadhyay and Sharma [25,26]. Well spread five metaphase plates were selected and photographed. Karyograms were drawn and lengths of long arm (L) and short arm (S) were measured for karyotype analysis. Karyotype analysis was carried out using Micro Measure Computer Program . Mean chromosome length (MCL) in μ, the total chromosome volume (TCV) and total chromosome length (TCL) were determined. To estimate karyotype asymmetry, two numerical parameters, namely intra-chromosomal asymmetry index (A1) and inter-chromosomal asymmetry index (A2) were used according to Zarco . Symmetry percent (S%), resemblance between chromosomes (Rec. index), the symmetric indices (SYI index) and total form percent (TF%) which is the average degree of symmetry over the whole karyotype were calculated according to Huziwara .
The study confirmed existence of remarkable degree of chromosome variability among the three species. Near sub-metacentric (nsm) and near metacentric (nm) chromosomes were common in both karyotypes (Figures 1-3) (Tables 1-4). Idiogram of the haploid complements is shown in Figures 1-3 for T. alexandrinum, T. refeigratum and T. repens, respectively. The evolution of karyotype is estimated by indices of symmetry. These values theoretically ranged from 0 to 100 for Rec and Syi indices and from 0 to 50 for both TF% and symmetry (S%). A karyotype with high indices is considered as evolving slowly .
|Chromosome pair number||Chromosome length(µm)||Arm Ratio||Centromere Index||S/L||Karyotype|
|Length each||Long arm||Short arm||(L/S)||(S/(L+S))|
Nsm = nearly sub-metacentric; nm = nearly metacentric
Table 1: The average measurements and arm ratios of somatic chromosomes of T.repens.
|Chromosome length (µm)||Arm Ratio||Centromere. Index||S/L||Karyotype|
|Length each||Long arm||Short arm||nsm(-)|
*nsm = nearly sub-metacentric; nm = nearly metacentric
Table 2: The average measurements and arm ratios of somatic chromosomes of T. refeigratum.
|Chromosome pair number||Chromosome length (µm)||Arm ratio||Centromere. Index||Karyotype|
|Length each||Long arm||Short arm||(L/S)||(S/(L+S))||S/L|
nsm = nearly sub-metacentric; nm = nearly metacentric
Table 3: The average measurements and arm ratios of somatic chromosomes of T. alexandrinum.
|No.||Species||TCL(µ)||MCL(µ)||S%||TF%||A1||A2||Syi index||Rec index||Karyotype|
|± SE||± SE||formula|
|1||T. alexandrinum||110.5||6.9||0.15||44.81||0.53||0.21||0.31||0.75||2 nsm(+),10 nsm|
|± 1.46||± 0.16||(-)+4 nm|
|2||T. refeigratum||227.9||7.12||0.15||48.55||0.52||0.21||0.32||0.65||4 nsm(+),24 nsm|
|± 1.47||± 0.14||(-)+4 nm|
|3||T. repens||262||8.19||0.128||53.53||0.48||0.27||0.33||0.64||8 nsm(+),12 nsm|
|± 2.20||±0.19||(-)+12 nm|
*Mean chromosome length (MCL) in μ, the total chromosome volume (TCV) and the total chromosome length (TCL) were determined. To estimate karyotype asymmetry, two numerical parameters, namely intra-chromosomal asymmetry index (A1) and (inter-chromosomal asymmetry index (A2), Symmetry percent (S%), resemblance between chromosomes (Rec. index), the symmetric indices (SYi index) and the total form percent (TF%).
Table 4: Karyotype parameters of somatic chromosomes of three species of Trifolium.
Most species of Trifolium are diploid (2n = 16) and only 16% of the 248 species are polyploidy . About 70% of the known polyploids occur in the subgenus Amoria, which is considered to be the most primitive and unspecialized subgenera.
Both T. refeigratum and T. repens have16 pairs of chromosomes with a pair of satellites located at the end of their short arm in chromosome 16, confirming the earlier results reported by Chen and Pryce  (Figure 4). Somatic chromosome karyotype was constructed from 11 mitotic cells by arranging the chromosome pairs on the basis of decreasing size and centromere position, with classification on the basis of the arm ratio using the criteria of Levan et al. .
Karyotype analysis data from Table 1 and Figure 1 revealed that the 11 pairs of chromosomes were nearly sub-metacentric [(nsm) (-)] (chromosomes 1, 2, 3, 4, 7, 8, 9, 11, 12, 13, 14, and 15) and 4 pairs of chromosome were nearly meta centric (nm) (chromosomes 5, 6, 10 and 16). Chen and Pryce  in a less detailed analysis, reported that 4 pairs of the T. repens chromosomes were meta centric, 11 pairs submeta centric, and 1 pair telocentric. Furthermore, T. alexandrinum has 8 pairs of chromosomes with karyotyping formula is 2 nsm (+), 10 nsm (-) + 4 nm.
Egizia et al. found that the changes in chromosome number have played an important role in the evolution of the genus Trifolium . Along with a few species of polyploidy origin, there are several cases of diploid as evidenced by the presence of four basic chromosome numbers (x = 8, 7, 6, 5). T. subterraneum and T. israeliticum are related species with chromosome complements of 2n = 16 and 2n = 12, respectively. Although they represent an interesting case of speciation based on chromosome number reduction, no attempts to demonstrate their cytogenetic affinity have been carried out so far. The present study performed a comparative cytogenetic study with the purpose of clarifying the evolutionary relationship between these species and to verify whether genomic rearrangements, other than modification of the chromosome number, are associated with the speciation process. Although karyo-morphological analysis supports the hypothesis that chromosome rearrangements had a role in the reduction of the chromosome number, the physical mapping of the rDNA sequences revealed a significant re-modeling of the 45S and 5S rDNA sites that greatly contributed to the differentiation of the 2n = 16 and 2n = 12 karotypes. The nucleotide analysis of 5S rDNA repeats confirmed that the two species are related but distinct. The observed genomic changes lead to the hypothesis that the 2n = 12 species is the result of an evolutionary pathway that passed through intermediate forms. It cannot be excluded that the most direct ancestor of T. israeliticum was a species with 2n = 14.
From Table 4 note the contrast among the three species and Egyptian clover in different values along the chromosome in total length, as well as describing the average length of the chromosome. Egyptian clover has equal T. refeigratum ratio in the S% while differed from them in white clover (T. repens). Also, TF% was 44.81 in Egyptian clover (T. alexandrinum) while values in T. refeigratum and T. repens were 48.55 and 53.53, respectively. These results reflect the size of the chromosome and the length of the chromosome in each species. Furthermore, values in A1 and A2 that give the duplication in T. alexandrinum was diploid and tetraploid in T. refeigratum and T. repens.
The data in (Table 5 and Figures 5 and 6) revealed that the T. alexandrinum was clustering alone and has polymorphic bands that differ from T. refeigratum and T. repens. Kumar et al. and Lange and Schifino [34,35], who studied Trifolium species and, related the narrow genetic base of Trifolium species due to incompatibility barriers. Isozyme variation in wild and cultivated species of the genus Trifolium was noticeable among eight Trifolium species. Malaviya and Rao  evaluated some lines of T. alexandrinum for pollination behavior, morphology and yield. Biochemical markers, especially the electrophoretic profiles of isozymes and proteins, have been widely used for identification of cultivars. They find that electrophoretic methods have been standardized for a large number of crops and were found useful for the purpose of Indian cultivar identification and characterization. It is shown that the two clover lines as well as their plasma radiation treatments had different protein profiles, which reflects their genetic diversity.
|MW (KDa)||T. repens||T. refeigratum||T. alexandrinum|
Table 5: Polymorphism among three species of Trifolium based on seed storage protein.
Karyotype analysis and protein profile can explore taxonomy via polyploidy and genetic distance among three species of Trifolium, T. alexandrinum have 8 pairs of chromosomes whose karyotyping formula is 2 nsm (+), 10 nsm (-) + 4 nm. The T. alexandrinum was clustering alone as well as having polymorphic bands that were different from the two other species T. refeigratum and T. repens.
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